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Introduction

The ultimate success of strategies to prevent sexual transmission of HIV depends, in part, on a better understanding of the virological and immunological mechanisms that define sexual transmission [1]. Our understanding of HIV-1 shedding in the female genital tract has increased greatly since the publication of the last AIDS comprehensive reviews of HIV shedding in the genital tract [2,3]. Here we review the literature published over the last 5 years, including virological, microbiological and immunological parameters that affect HIV-1 pathogenesis and transmission. Although several biological and behavioral factors influence the sexual transmission of HIV, this review focuses on the genital and systemic factors that define the infectiousness of HIV and susceptibility to sexual transmission [4,5]. Throughout, we compare HIV-1 shedding in the genital tract of women versus men.

Evaluation of virological and immunological parameters in the genital tract

HIV was first isolated from the genital tract of men [6,7] and women [8,9] approximately 15 years ago. Between 1988 and 1995, only 15 and five reports, respectively, described detection of HIV in the genital tract of men and women [2]. Additionally, there were few confirmed correlates of HIV-1 shedding in the genital tract for either men or women, although more was known about men [2].

Female genital tract

Anatomical considerations and source of HIV shedding

Although HIV can be recovered from the vagina of women who have had a total hysterectomy, most genital virus arises from the cervix and possibly the upper genital tract [10–14]. The proximity to the vaginal lumen of cervical-stroma lymphocytes, which compose the genital-associated lymphoid tissue, probably contributes both to HIV shedding and to susceptibility to mucosal transmission. Moreover, there are important histologic changes in the lower reproductive tract over a woman's reproductive life that influence the choice of sampling methods to evaluate HIV shedding.

The cervical surface epithelium and underlying stromal inflammatory-cell constituents of the endo- and exocervix change throughout a woman's lifetime as a result of hormonal, physical and infectious influences. The squamocolumnar junction (Figs 1 and 2a) may lie exposed on the surface of the exocervix at menarche (ectropion, Fig. 2b), in most adolescent women and in almost all women following parity [15]. The squamocolumnar junction recedes up into the endocervical canal during the reproductive years and has completely receded by the time of menopause. This recession occurs by replacement (metaplasia) of the ectropion columnar epithelium with squamous epithelium to form the biologically important epithelial transformation zone (Fig. 2c). Little is known about the relationship between HIV shedding or susceptibility to HIV infection and the ectropion, epithelial transformation zone and phase of a woman's reproductive life cycle. However, the presence of ectopy has been associated with an increased risk for heterosexual transmission of HIV [16,17], and cervical inflammation has been associated with increased cervical shedding of HIV [18].

Estrogenic stimulation causes vaginocervical stratified squamous epithelium to proliferate and completely mature, whereas endocervical columnar epithelium reacts by increased mucin production. In theory, both responses to estrogen should strengthen the physical barrier to HIV infection during the follicular phase of the menstrual cycle. Progesterone, on the other hand, causes thickening of intermediate layers but does not lead to complete maturation of the epithelium. Interestingly, viral shedding from the female genital tract is lowest during the follicular phase and rises during progesterone stimulation of the luteal phase (as discussed below). Two recent studies have shown that neither oral nor injectable hormonal contraceptives are associated with an increased prevalence of ectopy [15,19].

Physical trauma to the exocervix as a result of sexual intercourse, tampon use, douching and infection facilitates epithelialization (epidermalization) of the ectropion [15,20,21]. Patients with cervicitis due to Chlamydia trachomatis may have superficial epithelial ulcers in both columnar and metaplastic epithelium. Cervical C. trachomatis infection is characterized histologically by a patchy periglandular inflammation composed of neutrophils and plasma cells, with an organization of activated stromal lymphocytes into discrete germinal centers below the epithelial basement membrane (Fig. 2c, panel iv) [20]. This inflammatory pattern differs from the deeper necrotic ulceration and stromal lymphocytic infiltrate associated with herpesvirus infection. Presumably, the presence of activated lymphocytes and organized lymphoid aggregates in such close proximity to HIV-infected seminal ejaculate in the endocervical lumen or vaginal vault will increase susceptibility to HIV infection among women with sexually transmitted infections (STIs). Conversely, HIV-infected women with STIs are expected to have increased shedding of HIV from the genital tract, increasing their potential infectiousness (as discussed below) [18,22].

Sampling of the female genital tract for HIV

Sampling methods for the female genital tract have traditionally involved cervicovaginal lavage (CVL) with sterile saline [23–26] or swabs of the cervix [26–29]. More recently, sampling methods have included direct aspirates of cervical mucous [24], cytobrush [25,28,30], tampons [31], swabs [29,32] and Sno-strip® (Chauvin Pharmaceuticals Ltd, Essex, UK) wicking [30,32] (Fig. 3). The advantage of the Sno-strip® wick is that it allows the localization and collection of a defined volume of genital fluid from a specific anatomical location such as the vaginal fornix, endocervix or exocervix with little or no disruption of the epithelial surface.

The moistened Weck-Cel® Sponge (Windsor Biomedical, Newton, New Hampshire, USA) has also been used for sampling mucosal antibodies [33–35]. As with the Sno-strip®, the Weck-Cel® Sponge is an ophthalmic fluid collection device that has been adapted for collection of cervical secretions [34,35].

These collection techniques permit the concentration of virus or antibodies and the detection of lower levels of virus or antibodies than might be identified in the more dilute (1–10 ml of fluid) CVL. The collection devices used for antibody quantitation require pre- and post-collection weighing and additional sample processing, including elution, which involves detergents and protease inhibitors. The dilution factor can be most accurately calculated from the Sno-strip®, since a known volume (∼ 8 μl) is absorbed up to the shoulder of each wick strip (Fig. 3) [36].

All sampling methods offer advantages and disadvantages (Table 1). CVL samples have the benefit of sample volume and can be readily fractionated into cellular and cell-free components. Moreover, CVL can be performed easily in the clinical setting and yields 400 times the volume for virological or immunological analysis of either Sno-strip® or sponge collection methods. The disadvantage of CVL is that virus and antibody levels will be diluted, and volume standardization is problematic. The volume can be standardized by adding 10 mmol/l lithium to the washing buffer [37,38]. This procedure has been tested on spiked specimens but has not been routinely applied to clinical samples. Swabs collect both cellular and cell-free material, as do direct aspirates. In contrast, cytobrushes collect primarily cell-associated material (epithelial cells and inflammatory cells present on the mucosal surface), while Sno-strips® and the Weck-Cel® Sponge collect primarily cell-free material. Direct aspirates present problems in volume standardization, similar to those for CVL.

The variability of assay results is a function of the sampling method, assay and biological variation in the compartment sampled [39]. Variability in female genital tract measurement parameters has been determined only for HIV-1 RNA level. Variability is least for Sno-strip® wicks and greatest for CVL samples [30]. Variability is similar for Sno-strip® wicks and cytobrushes, and within-subject variability approximates ± 1.1 log10 RNA copies/ml (11-fold); this variation is greater than that for blood plasma HIV-1 RNA, which is two- to seven-fold [30,36,40]. The Sno-strip® wick is the optimum method for collecting a known volume of sample; however, the sample volume is quite small, which limits the use of the specimen to one assay.

Because more than one sampling method may be used in complex studies, the order of sampling becomes important. Depending on the assay, sampling by one method may actually enhance sampling by another method [26]. In general, the approach to sampling order has been to proceed from the least intrusive to the most intrusive method [30]. Detection and quantitation of virus is greatest in the cervix [10] and least in the vagina [12,25,28,30,32].

Male genital tract

In contrast to the lower female genital tract, the male genital tract is inaccessible to simple, direct sampling. Therefore, the detection and quantification of HIV-1 in semen have largely defined our knowledge of HIV in men.

Semen includes cellular and cell-free components in a complex fluid matrix that varies with each ejaculate. The cellular components include sperm, polymorphonuclear cells, immature germ cells, lymphocytes, macrophages and occasional genitourinary epithelial cells (Fig. 4). Seminal plasma is made up of glandular secretions from the testes, epididymides, prostate, seminal vesicles, proximal bulbourethral glands and more distal periurethral glands. The major components of seminal fluid come from the prostate and seminal vesicles. Consequently, the precise composition of semen depends on physiological conditions and frequency of ejaculation.

The composition of the seminal ejaculate thus influences the biological properties of semen and contributes to an enormous between- and within-subject variation in mononuclear cell count, potential CD4+ cell targets for HIV, secretory fluid composition, viral RNA levels and, likely, the infectiousness of the HIV in seminal fluid (as discussed below). As in the vagina, cervix and uterus, the dynamic secretory function of the male genital tract must be considered when evaluating HIV dynamics and antiretroviral drug pharmacokinetics in semen [41].

Although T lymphocytes and macrophages in semen harbor HIV, there is still no convincing evidence that spermatozoa are infected with virus [42] (reviewed in [43]) (Fig. 4). However, sperm may potentiate HIV transmission by binding with virus to HLA-DR-positive cells, possibly through binding of env gp120 with sperm-associated galactoglycerolipid [44,45]. Nevertheless, the precise location of the HIV-producing genital cells has not been identified, and the contribution to transmission of cell-associated and cell-free virus in semen remains largely undefined, even though both forms of virus are transmissible [46] (the literature before 1995 is reviewed in [1]). Knowledge about the precise location and source of HIV in semen is critical for designing effective assisted reproductive technologies for HIV-discordant couples [47–56].

Several studies have confirmed earlier reports that HIV levels in semen are lower and much more variable than those in peripheral blood [57–60]. In a rhesus macaque model of chronic simian immunodeficiency virus (SIV) infection, Miller and colleagues used in situ hybridization and immunohistochemical staining to determine that SIV-infected T cells and macrophages are found at all levels of the reproductive tract, most consistently in the epididymis [61]. The same level of anatomical localization has not been reported for humans [43]. HIV is detected in the ejaculate of vasectomized, HIV-seropositive men and in pre-ejaculatory fluid, which suggests a more distal source of HIV [62–65].

Measurements of genital tract virus

HIV RNA quantification

Several studies have evaluated HIV-1 RNA assays from different manufacturers, and in general there is consensus among assays [66,67]. HIV-1 RNA can be measured as cell-associated or cell-free virus [25,32,46, 58,60,68–76]. In general, for women, HIV-1 RNA levels are highest in swab, cytobrush and Sno-strip® samples and lowest in CVL samples [25,26,30,32].

The ability to detect HIV-1 RNA in the female and male genital tracts is a function of the number of samples obtained [30,36,40,59]. Longitudinal studies lasting from 8 to 10 weeks have shown that viral RNA is detected continuously in 28–37% of seminal plasma samples, intermittently in 39–44% of samples and never in 24–28% of samples from HIV-infected men [58,59]. A longitudinal study over two menstrual cycles showed that genital viral RNA was detected continuously in 29% of samples, intermittently in 58% of samples and never in 13% of samples [30].

In general, viral RNA levels are higher in blood than in the corresponding genital fluids from men and women. However, compared to blood plasma, increased HIV RNA levels have sometimes been found in endocervical fluid [36] or CVL [70,77,78] and in semen [58,79].

A technical issue particular to semen is the presence of inhibitors of reverse transcriptase polymerase chain reaction (PCR) amplification in 40% or more of men [58,81]. Although the mechanism for this inhibition is unknown, semen contains several antimicrobial compounds that specifically inhibit some viral enzymes; this inhibition can be removed by pre-treatment with silica gel or commercial elution reagents [58,67,81]. In contrast, no inhibitors of HIV PCR were detected in spiked CVL samples obtained from HIV-uninfected women over the menstrual cycle [82].

HIV DNA detection

Proviral DNA copy number from cervical and vaginal swabs has also been assessed [29,73,83]. In the Mostad study [29], HIV-1 DNA was detected more frequently in endocervical versus vaginal swabs (46 versus 17%), although detection was quite variable for any single woman. Detection by either method was correlated with detection by the other method and with blood RNA levels.

Similar associations between semen and blood HIV-1 DNA have been reported [80,84,85]. In a study of 34 HIV-1-infected men, proviral DNA was detected in 100 and 47% of paired blood and semen samples, respectively, and viral RNA was detected in 76 and 63% of paired samples, respectively [84]. In another study of 52 HIV-1-infected men, HIV DNA was detected in 57.1% of non-spermatozoal cell fractions. By comparison, HIV RNA was detected in 86.5% of seminal plasma specimens and 14.6% of spermatozoa fractions.

HIV culture

The frequency of detection of culturable virus, either cell-free or cell-associated, is low in the female genital tract, even in older studies conducted in the absence of antiretroviral therapy (ART) [8,9,86]. More recently, virus has been cultured from direct aspirates, swabs, CVL cell pellets and cytobrush samples [24,26,70, 71,83]. The collection methods have not been found to differ in terms of the culturability of virus [26,70]. The frequency of culturable virus is not only quite low (5%), but at a level too low to be quantified accurately [71]. However, as with HIV-1 RNA detection, the number of positive cultures increases with sampling frequency; thus, up to 20% of women may be culture positive if sampled frequently enough (unpublished data).

HIV is cultured more often from the male genital tract (20–50%) than from the female genital tract. Most of the culturable virus is recovered from the cellular fraction (∼ 90%) and less from the cell-free, seminal plasma (∼ 10%). Quantitative and qualitative HIV culture of seminal cells correlates with HIV RNA levels in semen [58,80,87].

Genital tract immunological measurements

Female

Procedures used to evaluate immune parameters in the female genital tract have included quantification of antibody levels and cytokines in genital tract secretions as well as evaluation of cellular immune elements. The most complete studies have evaluated antibody levels in the genital tract and compared the Sno-strip® wick and CVL collection methods [88]. Cytobrush has also been used to obtain cervical cells for cytokine quantification [89,90]. The results of numerous studies using various collection methods have demonstrated that IgG antibody to HIV predominates in the female genital tract, with low to undetectable HIV-specific IgA antibody [91,92]. Additional studies have found that the IgG1 and IgG3 subclasses predominate in cervical secretions [93]. These subclasses have been associated with functional activity of antibody-dependent cellular cytotoxicity and complement-activating activity. Recent studies have shown that the presence of antibody-dependent cell-mediated cytotoxicity in CVL samples from HIV-infected women correlates with CVL viral load [94].

Techniques for obtaining immune cell subsets from the female genital tract are neither well-developed nor standardized. In most studies, either CVL or cytobrush collection techniques were used, but quantitative comparisons between the two methods should be interpreted with caution because of the variability in cell type, cell number and viability [90].

Endocervical sampling has been shown to produce the highest cellular yields consisting of T cells, B cells and monocytes [95]. CD4+ and CD8+ T cells can both be found, with CD8+ cells in preponderance. As yet, there is no clear correlation of HIV disease stage, or therapy, and ability to isolate these immune cells. However, studies have demonstrated functional HIV-specific activity of CD8+ cells obtained in this fashion [89,90].

Additional cellular studies have relied on tissue biopsies from the female genital tract. CD4+ and CD8+ T cells have been detected in uterine, cervical and vaginal tissue biopsies [96]. Isolated populations from the cervical mucosa have demonstrated slightly more CD8+ than CD4+ cells, but both populations have a predominant memory phenotype (CD45RO+ CD45 RA−) and express the chemokine receptor CCR5 [97].

Increased levels of CD4+ memory cells have been found in cervical mucosal biopsies in HIV-negative subjects with underlying genital tract infections. The finding of a predominance of CCR5 expression on these cells might be an important factor for the predominant transmission of HIV R5 strains heterosexually [98].

Male

Evaluation of immune parameters in the male genital tract has consisted of studies of immunoglobulins and cytokine levels and immune cellular elements in semen [99–101]. Because the level of antibodies and cytokine in semen is quite low, more sensitive assays are required to make accurate measurements. In addition, various factors in semen, including proteases, can affect the integrity of these analytes, and samples must therefore be processed rapidly. Because immune cells (lymphocytes and monocytes) are present at low numbers in semen, cell analysis methods such as flow cytometry are not readily applicable [99] (Fig. 4). Additional methods must be developed to accurately evaluate seminal cellular constituents.

The level of total IgA in semen appears to be lower than total IgG levels in both HIV-positive and HIV-negative men [102]. However, there is no evidence of local production of IgG or IgA. gp120 and p24 HIV-specific IgG antibodies are found in most semen samples, but lower levels of HIV-specific IgA antibody are seen [102].

Cytokine levels have been quantified in semen, and these studies showed that tumor growth factor β (TGF-β), interleukin-8 (IL8) and IL6 can be detected in seminal samples from both HIV-positive and HIV-negative men [99]. Interferon γ (IFNγ) can also be detected in semen. Evaluation of cellular elements in semen has shown the predominance of CD8+ T cells. In one study, CD8+ T cells were shown to have HIV-specific cytotoxic activity [103].

Measurement variability

Few studies have evaluated measurement variability in immune analytes, including antibody and cytokine measurements from the female genital tract. The most extensive analysis used the Weck-Cel® Sponge to obtain sufficient undiluted material for multiple measurements [104]. The analytes evaluated included total IgG, IgA, IL12 and IL10. The study demonstrated no significant difference in duplicate samples obtained from the same woman (Spearman correlation 0.78–0.84). The only cytokine with poor assay reproducibility was IL10; however, the authors attributed this low reproducibility to the IL10 assay itself and not the collection method.

Similar evaluations of immune analyte measurement variability have not been done for men.

Correlates of HIV-1 virus shedding in the genital tract

The most common genital co-infection in HIV-infected women and men is human herpes simplex virus type 2 (HSV-2). The worldwide prevalence of genital herpes infection suggests that HSV increases the efficiency of HIV-1 transmission and has a negative impact on the natural history of HIV (reviewed in [105]).

The biological basis for implicating HSV as an important cofactor in HIV transmission and pathogenesis involves more than just the disruption of normal epithelial barriers to HIV infection (Fig. 2). The strong association between HIV, HSV-2 and male circumcision suggests that differences in the efficiency of HIV transmission are primarily mediated by biological factors that may outweigh differences in sexual behavior [106]. In addition, the increased shedding of HIV from HSV ulcers and the biological modulation between HSV and HIV have been demonstrated (reviewed in [107]).

Not surprisingly, HIV also shows a significant increase in replication in the presence of HHV-8 in in vitro and in vivo models. Although the mechanism responsible for HHV-8 induction of HIV-1 replication remains to be identified, evidence suggests that these two viruses may interact at the molecular level in co-infected patients by mechanisms similar to those of the other herpesviruses [108].

Microbiological correlates for women

The epidemiological interactions between sexually transmitted infections (STIs) and HIV in terms of susceptibility and infectiousness have been extensively reviewed elsewhere [109,110]. In general, genital ulcerative disease increases susceptibility to HIV infection more than does non-ulcerative disease and the impact of genital ulcerative disease is greatest for men. Although HIV infectiousness from men to women is increased by the presence of STIs, it is not clear that the same holds true for transmission from women to men.

Gynecological infections associated with HIV infection in women have also been extensively reviewed [111]. In many instances, the severity and frequency of STIs is enhanced by HIV infection. Although epidemiological studies provide some insight into the interaction of HIV and concurrent genital infections, these studies have not directly assessed the interaction between HIV and concurrent genital infections in vivo, and the results may differ depending on the patient population. For example, in US populations, human papillomavirus (HPV) and associated neoplasms were more prevalent among HIV-1-infected women [112–116], as were genital ulcers and warts, vaginal candidiasis [117], HSV-2 [118] and C. trachomatis [119,120] (reviewed in [111]). In contrast to US populations, there was no overall association between HPV and HIV in Tanzania [121]. Additionally, increased HIV prevalence has been associated with bacterial vaginosis [122], antiseptic douching [114], hormonal contraceptive use [123] and some sexual practices [124,125].

The rationale for increased genital HIV-1 shedding in the presence of genital co-infections may seem apparent (Fig. 2), that is, the recruitment of increased numbers of infectable target cells and immune upregulation at the site of infection (as described below). However, reports of microbiological correlates of HIV-1 shedding in the female genital tract have often been in disagreement. These differences can be attributed in part to study design (cross-sectional, longitudinal, or therapeutic) and to differences in study populations. Additionally, association with the microbiological correlate may vary with the outcome measure (e.g., HIV-1 proviral DNA vs. cell-free HIV-1 RNA). With these caveats, Table 2 updates the review by Rotchford et al. [120] and describes some of the significant (P ≤ 0.05) correlates of HIV-1 shedding in the female genital tract in studies published in the last 6 years. Previously, only pregnancy, hormonal contraceptives, cervical ectopy and cervicitis had been associated with a significantly increased risk of HIV-1 detection in the female genital tract [2].

Validating studies of association is difficult. For example, among studies that have assessed the association between C. trachomatis infection and HIV shedding in African populations, no increase in shedding was seen in two cross-sectional studies that used HIV DNA as an endpoint [126,127]. In contrast, a significant increase was seen in a larger study that used HIV RNA as an outcome measure [128]. In subsequent meta-analyses, whether or not men were included, an association between C. trachomatis infection and HIV shedding could be shown [119,120].

The more convincing data on the effect of local co-infection with other pathogens on increased HIV shedding in the genital tract are derived from treatment studies in which genital viral loads are monitored pre- and post-treatment in HIV-infected women who present with a co-pathogen. In the C. trachomatis example, viral RNA levels decreased following treatment of C. trachomatis, but viral DNA levels did not [129]. This suggests that co-pathogens may differentially affect cell-free and cell-associated HIV shedding.

The majority of STI treatment studies have indicated that treatment of most genital co-pathogens results in a reduction of HIV-1 genital tract viral load. An exception was the treatment study of bacterial vaginosis by Wang et al. [130]. Randomized clinical trials are needed to determine the relationship between treatment of various co-factors to reduce genital HIV virus load and subsequent reduction in transmission.

Only limited recent data are available for cytomegalovirus (CMV), HHV-8 and HIV genital shedding. The prevalence of CMV shedding from the cervix was 19.6% in populations of CMV/HIV co-infected women before 1996 [131]. In one recent study, asymptomatic shedding of CMV DNA was common over a single menstrual cycle in 17 HIV-infected women with CMV antibody [132]. HHV-8 seroprevalence was relatively low (15%) in a large cohort of HIV-infected women [133]. Other data suggest a similar prevalence of these two potential viral cofactors in HIV-infected women [70,134–136]. The presence of both herpesviruses in the genital tract of HIV-infected women requires a better assessment of CMV and HHV-8 as cofactors for genital HIV shedding.

In contrast to the observed reduction in HIV load following treatment of co-pathogens, treatment of genital ulceration apparently activates proinflammatory cytokines, which in turn may transiently increase the levels of HIV [137].

One of the more intriguing aspects of these correlates is their potential mode of action in enhancing the replication of HIV-1. In addition to immune modulation (discussed below), a soluble factor that increases expression of HIV-1 in human monocyte cell lines, HIV-inducing factor (HIF), has been observed [138,139]. Various organisms associated with bacterial vaginosis – including Mycoplasma hominus [140], specific biotypes of Gardnerella vaginalis [141,142] and Peptostreptococcus aschcharolyticus [143] – appear to be associated with HIF. This factor appears to be a myeloid-related protein [144] that upregulates HIV expression by activating a kappa-B enhancer of the viral long terminal repeat (LTR) while also stimulating AP-1-dependent transcription [145].

Microbiological correlates for men

Symptomatic and asymptomatic urethritis are important cofactors of HIV shedding in semen, suggesting that local genital tract infections are important determinants of HIV levels in semen [148,154]. In one study, the best multivariate model for predicting HIV-1 culture positivity from semen included two local urogenital factors: increased seminal polymorphonuclear cell count and a positive CMV co-culture. The best multivariate model for predicting HIV-1 RNA detection from semen included CD4+ cell counts < 200 × 106 cells/l, nucleoside ART and a positive seminal CMV co-culture [147].

An independent association has been found between the level of cellular inflammation and HIV replication in vitro [155] and HIV shedding in vivo [147]. For example, antibiotic treatment of gonococcal urethritis reduces viral shedding in semen but not blood [148,153]. Few prospective studies have evaluated the effect of STI treatment on HIV shedding [120]. Importantly, effective ART appears to limit the effect of urethritis on seminal viral RNA levels; however, when blood plasma viral RNA levels are poorly controlled, high seminal viral RNA levels have occurred during gonococcal urethritis, increasing the potential for sexual transmission [153].

Taken together, these studies and the vasectomy study of Krieger et al. [64] strongly suggest that a primary site of HIV production may be the male urethra (i.e., from the periurethral glandular stroma, as suggested by other investigators [63,65]). Further studies are clearly required to definitively evaluate this possibility.

Immunological correlates for women

Many underlying infections in the female genital tract, including bacterial vaginosis and sexually transmitted pathogens (HPV, C. trachomatis, Neisseria gonorrhoeae), can lead to an upregulation of the proinflammatory [IL1β, IL6, tumor necrosis factor α (TNFα)] and immunoregulatory (IL12, IL10) cytokines that modulate HIV replication [156]. Proinflammatory cytokines can directly stimulate HIV replication by activating the LTR sequence of HIV. This finding may be especially important in parts of the world where HIV clade C virus is predominant, as this clade has been shown to have additional LTR-binding sites for TNFα [157]. Several immunological correlates of HIV shedding are summarized in Table 4.

The immunoregulatory cytokines IL12 and IL10 are important for their primary regulation of cellular immune responses in addition to their secondary regulation of HIV replication. Elevated IL10 levels are associated with the presence of sexually transmitted pathogens [158]. IL10 suppresses IL12, which is important for activating HIV-specific cellular immunity and for upregulating CCR5 co-receptor expression, especially on monocyte/macrophage targets. In addition, type 2 cytokine mRNA in genital tract mucosa is increased in HIV-positive versus HIV-negative women [159].

A recent comparison of Thai versus US women demonstrated a correlation between inflammatory T cell infiltration in vaginal mucosa and high viral loads in cervical tissue from Thai women. This study has implications for the higher rate of heterosexual transmission among Thai women [160].

Immunological correlates for men

Studies that have correlated immune parameters and HIV in men have evaluated the cellular components that harbor HIV in semen. Semen contains CD4+ and CD8+ T cells as well as seminal macrophages. CD4+ T cells and macrophages have been found to harbor HIV DNA. In HIV-positive subjects, CD8+ cells constitute 75% of the T-cell fraction in semen. These cells express the lytic molecule TIA-1, which suggests that they have a potential anti-HIV function [101,103].

Other factors correlated with genital tract shedding of HIV

Increased HIV-1 RNA and DNA shedding in the genital tract has been seen in women with selenium [161] and vitamin A [127] deficiencies in some cross-sectional studies but not in others [162]. However, a recent randomized, placebo-controlled clinical trial failed to show an effect of vitamin A supplementation on plasma or genital levels of HIV [163]. Additionally, use of injected and ingested hormonal contraceptives has been found to be associated with cervical proviral detection [127]. The effect of either nutritional deficiencies or the use of female sex steroid hormones in treating gender dysphoria on HIV shedding in semen has not been determined.

Interactions between peripheral and genital compartments

Plasma RNA

The relationship between HIV-1 RNA in blood and the level of HIV-1 RNA in the male and female genital compartment has been extensively evaluated in cross-sectional [23,25,58,79,80,84,126,164–171] and longitudinal [28,30,70,118,172–175] studies. HIV-1 RNA can be detected in most male and female genital samples with incomplete plasma viral load suppression [58,70,77,79] and in approximately 20% of specimens from women with complete plasma viral RNA suppression [30,70,176]. All but two studies [118,171] have found an association between plasma viral RNA levels and the ability to detect virus in the genital tract.

The study by Mbopi-Keou et al. [118] points out the problem with studies that assess plasma RNA in isolation from other cofactors that determine genital HIV shedding. In this central African study population, 57% of subjects were positive for bacterial vaginosis, 41% for Candida and 43% for HSV; the latter was the strongest predictor of genital shedding.

Many studies assessing correlations between plasma HIV RNA and genital shedding have specifically excluded women with overt genital infections. One of the largest cross-sectional studies found that genital co-infections are secondary in importance to plasma viral load [70], whereas another study found that plasma viral load and bacterial vaginosis are independently predictive of HIV detection [175]. In two longitudinal studies, genital co-factors and plasma viral load were independently predictive of genital tract HIV detection [30,70]; each log10 increase in plasma viral RNA was associated with a three- to six-fold increase in HIV detection frequency in the genital tract.

The most convincing data on the association of plasma viral RNA with genital HIV level comes from treatment studies where women who initiated or changed ART were monitored for changes in genital viral load [25,172]. Women with reduced plasma viral loads had HIV detected less frequently in the genital tract. Whereas the correlation between viral RNA levels and genital tract detection of virus is good, up to one-third of women and less than 4% of men have undetectable plasma viral RNA but detectable genital tract RNA [58,70,76,118,175,177,178]. Local genital tract replication of HIV clearly occurs in men and in the endocervical/vaginal cavity of women and may contribute to this discordance [58,72,170].

Few studies have assessed the relationship of peripheral blood levels of culturable virus [70] or proviral DNA copy number [28,84] with genital viral load, assessed by either RNA or DNA levels. Although there is an association among these other viral parameters in blood and genital tract, plasma RNA level is a strong predictor of viral shedding from the genital tract. The greater variability in the other blood measurements may result in an underestimate of their contribution to genital levels of virus.

CD4 and other immune markers

Studies in men and women indicate that reduced CD4+ cell count is associated with increased immune dysregulation in the genital tract, as shown by increased shedding of HIV-infected cells and reduced ability to isolate cells with functional anti-HIV activity. Similarly to peripheral blood, there appears to be a reduced ability to detect HIV-specific CD8+ cells in the genital tract of subjects on ART [90]. However, the HIV specificity of cells in the genital tract differs from that of peripheral blood, indicating the compartmentalization of viral replication.

Some studies have demonstrated dysregulation of cytokine production within the local mucosal environment in subjects whose peripheral immune system is intact [179]. Several studies have also demonstrated that women who are highly exposed mucosally to HIV but remain uninfected will develop, in the genital tract, evidence of HIV-specific IgA antibodies with functional neutralizing ability [180–185]. These uninfected women also have HIV-specific CD8+ effector cells that can be isolated by cervical cytobrush collection [186]. Thus, the female genital tract may be an important site for inducing an immune response as a first line of defense against HIV transmission.

Antiretroviral therapy

The relationship between ART and genital tract shedding of HIV is complex and likely to become more so. Most of the studies reviewed in this paper were conducted when the therapy guidelines recommended that ART be initiated in patients with CD4+ cell counts ≤ 500 × 106 cells/l. For many reasons, women were less likely to receive therapy; even in recent studies, approximately 25% of women did not receive ART. In spite of the exceptions noted above, an overall association exists between ART and inability to detect virus in the genital tract [30,68,70,72,76,187, 188].

Large epidemiological studies have shown that plasma RNA levels are a major contributor to HIV transmission [189]; although studies are contemplated, no study to date has looked prospectively at the effect of plasma viral load reduction on reducing the sexual transmission of HIV. Interestingly, recent recommendations to postpone ART until CD4+ levels are < 350 × 106 cells/l may have a negative impact on the sexual transmission of HIV because of higher genital HIV levels in the sexually active, untreated HIV-infected population with CD4+ counts ≥ 350 × 106 cells/l.

Antiretroviral drug penetration into genital fluids

Peer-reviewed reports concerning antiretroviral drug penetration into the female genital tract were limited at the time of this review. In one study, protease inhibitors penetrated the female reproductive tract at suboptimal concentrations, but neither adherence nor the timing of the CVL sample relative to oral drug intake was accounted for [78].

In men, penetration into the semen is drug specific and depends on physiochemical properties of the drug and biological mechanisms that are host specific (reviewed in [190]). Studies have shown that HIV RNA levels are reduced in semen by ART [165–167,187,191], but whether this reduction in viral level will result in a concomitant reduction in sexual transmission of HIV is unknown. Moreover, because combination ART regimens have been used in these studies, it is unknown which drugs and drug concentrations are necessary to suppress genital tract HIV levels [190].

Antiretroviral drug resistance in the genital tract

Therapy that reduces virus in the blood to an undetectable level may not completely suppress virus in the genital tract of women [70] or men [3,191,192]. In a longitudinal study of men and women who had complete viral suppression for up to 2 years, virus was found in the semen of one of 21 men and in cervical secretions of two of two women [178]. Not surprisingly, as a consequence of ART, drug-resistant virus may be present in the female genital tract [78] and in semen [74,193–195] and is transmitted sexually between men [196–198]. Because of the increasing prevalence of virus with decreased drug susceptibility, additional studies are needed to identify the determinants of sexual transmission and drug-resistant virus in genital fluids.

Hormonal changes over the menstrual cycle and the impact on HIV shedding

Variation in HIV-1 genital tract shedding

HIV-infected women without AIDS appear to have normal menstrual cycles and normal levels of progesterone and estradiol [199,200]. Although sex-based differences in HIV infection are likely to be hormonal, the effects of endogenous and exogenous hormones on HIV pathogenesis are still poorly understood. Several studies have failed to identify an effect of the menstrual cycle on HIV detection in the genital tract [40,174, 201] or variation in HIV levels in blood [36,40]. However, one study found a specific association between the menstrual cycle and HIV-1 RNA levels in the genital tract [36], and another study found that ovulatory cycles were associated with a decline in blood HIV RNA levels [202].

The apparent differences among these studies may reflect anovulatory menstrual cycles, technical differences in specimen collection methods, genital site sampled, assay methodology and data analysis [201–203]. For example, many studies have relied on an evaluation of HIV RNA levels in CVL, while others have used endocervical swabs or wicks. The evaluation of viral RNA changes in CVL is imprecise because of the high within-subject variability in viral RNA levels associated with the CVL dilution step required for specimen collection. The total expected within-subject variability of HIV RNA levels is 3.5-fold higher in CVL than in endocervical canal fluid [30]. As such, studies relying on CVL sampling alone will miss small changes in viral RNA levels.

Genital secretions from the endocervical canal have been collected during all phases of the menstrual cycle with the more precise Sno-strip® wick [36]. The mean HIV RNA levels were not only greater in endocervical wick than in CVL (even when correcting for CVL dilution) but were significantly greater than viral RNA levels in blood plasma. This difference in endocervical wick HIV RNA peaked during the week before menses (P = 0.03). These findings have a biological basis, as proinflammatory cytokines in the vagina but not in blood plasma are also upregulated at the time of menses [204,205].

The same study also found that endocervical canal cytobrush sampling, with a variability between that of wick and CVL sampling, did not demonstrate the same association with the menstrual cycle as endocervical wick. This difference between cell-free endocervical fluid and cell-associated viral nucleic acid levels reflects not only technical differences in sample collection but also the compartment in which HIV RNA was measured.

A major concern in all these studies has been the contribution of blood-associated HIV to the vaginal measurement of virus during the menstrual cycle. This contribution is probably minimal [25,36]; as such, evaluations of the effect of the menstrual cycle on HIV-1 parameters should include sampling during menses.

Variation in HIV-1 co-factors

Few studies have assessed variation over the menstrual cycle for most of the co-factors present in the female genital tract. An increase in vulvovaginal candidiasis during the luteal phase of the menstrual cycle is assumed but has been documented in only a single study [206]. Studies of HIV-uninfected women have shown menstrual cycle variation for chlamydia cervicitis [207], Escherichia coli [208], Bacteriodes fragilis [209] and complications of Neisseria gonorrheoae infection [210]. Additionally, microflora changes over the menstrual cycle may be influenced by the use of tampons or sanitary napkins [211]. In HIV-infected women, no variation in HSV shedding was detected over the menstrual cycle, but a two-fold increase in CMV detection was observed during the luteal phase [132]; both viruses were detected by DNA PCR. Recent studies in HIV-infected women showed no changes in culturable herpesvirus, bacterial vaginosis (as defined by the Amstel criteria) or trichomoniasis over the menstrual cycle. However, candida colonization was more frequent during the luteal phase (Jonathan Cohn, MD, personal communication, 2002).

Variation in immunological parameters

Several studies have evaluated changes in immune cell subsets in the female reproductive tract during the menstrual cycle [13,96,212]. There is evidence that CD8+ cell numbers and cytokine function are under hormonal influence. CD8+ cells from the uterus of post-menopausal women had the highest activity and cells from pre-menopausal women had the lowest activity during the secretory phase of the menstrual cycle [96].

The cytolytic activity of CD8+ cells from vaginal and cervical tissue is independent of the menstrual cycle [213]. Additionally, T cell (CD4+ and CD8+) and B cell numbers in the vagina do not vary during the menstrual cycle. In contrast, expression of polymeric immunoglobulin receptors by cervical and uterine epithelial cells varies with the menstrual cycle [214], which may explain the influence of the menstrual cycle on humoral immune responses [215,216].

Fewer studies have evaluated the impact of the menstrual cycle on immune parameters in HIV-infected subjects. Changes in cytokine levels in the mucosal and systemic compartments during the menstrual cycle have been evaluated [205]. Cytokines – including IL8, RANTES (regulated upon activation, normal T-cell expressed and secreted), macrophage inflammatory protein 1β, TGF-β, IL10, IL4, IL6 and IL1β – were elevated in the genital tract with menses, but not during the other phases of the menstrual cycle. This elevation in cytokine levels also correlated with increased levels of HIV RNA detection in the genital tract. Compared to seronegative individuals, the only cytokine altered during the follicular phase of the menstrual cycle was IL6. The comparison of menstruation-related changes in the genital tract and peripheral blood has demonstrated that, except for IL6, most cytokine levels are regulated independently.

Confounding variables

Innate immunity and natural host defenses

Women

Epithelial cells in the lower female genital tract participate in immunological functions, their activity is upregulated by proinflammatory and immune cytokines and epithelial cell immunological functions vary at anatomical sites throughout the genital tract [217].

Studies of sex workers in Thailand and Africa have demonstrated that HIV-specific IgA predominates in the genital tract, and, in some subjects, systemic HIV-specific T-helper responses are seen [218]. These IgA antibodies have been shown to have neutralizing activity and an ability to block HIV transcytosis across an in vitro epithelial barrier [219].

In conjunction with the presence of HIV-specific antibody in CVL, increased levels of chemokine (RANTES) and type 1 cytokines (IFNγ) have been reported [220]. Other studies using cervical biopsies have demonstrated the presence of HIV-specific CD8+ cells capable of secreting IFNγ. This result is consistent with findings of HIV-positive CD8+ cytotoxic T lymphocytes in cervical samples from HIV-1-resistant female sex workers [186]. These studies demonstrate the existence of natural protection against HIV infection in the female genital tract. Similar studies of natural protection against HIV infection have not been carried out in men.

Several biological properties of the female genital tract provide protection from HIV-1 infection and potentially interact with each other to augment innate immunity to modify HIV-1 replication. These properties include a natural acidic pH, hydrogen peroxide-producing lactobacilli, mucous coating and a multi-layered epithelial barrier [221] (Fig. 2).

Antimicrobial peptides, a class of compounds found in insects, plants and animals, are natural microbiological inhibitors that target the microbial cellular membrane (reviewed in [222]). Much is known about the role of these compounds in blood [223] and the oral cavity [224] but not in the female or male genital tract. Although several of these molecules have been shown to have anti-HIV activity in vitro (i.e. integrins [225], defensins [226], protegrins [227] and secretory leucocyte protease inhibitors (SLPIs) [224,228,229]), the interaction between these molecules and HIV-1 in the genital tract has not been studied.

In vitro assays have shown that cervical tissue can produce peptide defensins [230,231] and SLPIs [217]. The inter-relationships between these antimicrobial peptides and other vaginal co-factors are potentially quite complex. For example, low vaginal pH in the presence of Lactobacillus species inhibits lymphocyte activation and contrasts with the lymphocyte activation seen with the higher pH characteristic of bacterial vaginosis [232]. Hydrogen peroxide-producing Lactobacillus species inhibit HIV-1 in vitro, which contrasts with the lack of HIV-1 inhibition seen with Lactobacillus species that do not produce hydrogen peroxide [233]. SLPI levels in vivo are decreased in women who have bacterial vaginosis with and without concurrent yeast vaginitis [234]. Moreover, hormonal contraceptives and the menstrual cycle (pre- versus post-menopausal) may modulate the production of antimicrobial peptides [230,235].

The epithelial layer from which these molecules arise in non-human primates is modulated by hormones [236]. However, in humans the modulation of the genital epithelial layer does not appear to be under a similar level of control [237].

In earlier reports, human chorionic gonadotropin purified from urine demonstrated activity against HIV [238]. However, this activity was due to the presence of contaminating urinary antiviral lysozyme and RNases [239].

The complex interaction between the HIV gp120 envelope protein and Candida albicans has been suggested as a mechanism by which HIV may promote the virulence of C. albicans in HIV-infected women [240,241]. Clearly, other studies of innate immunity in HIV-infected and uninfected individuals are needed.

Men

The anti-HIV properties of semen have not been well characterized. Semen does have numerous antiviral properties that may influence the survival of HIV-1 in the ejaculate and hamper the recovery of infectious virus in mixed lymphocyte culture [242]. For example, zinc, which is found in high concentrations in semen, is known to inhibit viral enzymes. Lactoferrin, an iron-binding glycoprotein present in many biological secretions, including semen, has anti-HIV-1 activity [243]. The polyamine spermine is found in exceptionally high concentrations in semen, and the polyamine-amine oxidase-peroxidase system inactivates a laboratory strain of HIV-1 in vitro [244]. SLPI is found in seminal plasma and has anti-HIV activity through targets on host cells rather than the virus itself [245] (reviewed in [246]). A seminal plasma CD4 ligand, the gp17 glycoprotein, which may function as an immunomodulatory CD4-binding factor at insemination, also inhibits HIV-1 envelope-induced syncytium formation in vitro and may play a role in controlling HIV spread in the genital tract [247].

Consideration must also be given to seminal plasma's viral and immunomodulatory properties. First, polyamines and deoxyribonucleoside triphosphates in seminal fluid may enhance natural endogenous reverse transcription of extracellular virions and thus facilitate the infectivity of nondividing target cells [248]. Second, seminal fluid must possess some degree of immune modulation to assure sperm survival in the female reproductive tract and may, as a consequence, potentiate the survival of HIV-1 [249]. The levels of prostaglandin (PG) in human semen are many orders of magnitude higher than those found elsewhere in the body, and semen contains 19-hydroxy PG E, which has not been found in other tissues [250]. The powerful effects of PG E and 19-hydroxy PG E on the balance of cytokines (stimulating IL10 and inhibiting IL12) released by antigen-presenting cells potentiates the survival of both sperm and HIV-1 by non-specifically inhibiting cell-mediated defenses [251]. In contrast, PG E2 may induce resistance to HIV-1 infection in monocyte-derived macrophages by downregulating the CCR5 co-receptor [252].

Phenotypic and co-receptor use

Only a few studies have evaluated genital tract viral tropism [72,253,254] and co-receptor use in the female genital tract [255,256]. Polymorphisms of CCR5 have been shown to result in increased levels of vaginal HIV-1 DNA but not HIV-1 plasma RNA or cervical DNA [28]. Ex vivo studies suggest several mechanisms by which R5 and X4 viruses can infect mucosa-associated CD4+ cells [255,256]. The hypothesis is that increased viral shedding is associated with an increase in CCR5 expression. However, it is unclear why this receptor polymorphism would be reflected in the vaginal but not the endocervical compartment.

In men, little is known about the contribution of viral phenotype to HIV transmission. Both syncytium- (SI) and non-syncytium- (NSI) inducing phenotypes (R4 and R5, respectively) are found in semen, but there is no signature-specific HIV envelope gene for semen [58,59,257,258]. In one study of 22 men, seminal HIV-1 isolates were phenotype concordant with blood for 16 men, or 73% (four SI and 12 NSI) and discordant for six men, or 27% (three SI and three NSI) [58].

If selective transmission of R5 isolates occurs, it is likely to occur in the recipient [3]. Although R5 virus may be found in the early stages of infection, it is not known whether R5 virus is selectively transmitted. Mononuclear cells that infiltrate the female genital mucosa are permissive for transmission of R5- and X4-tropic HIV-1 variants, and selection of virus variants does not occur by differential expression of HIV-1 co-receptors on genital mononuclear cells [255,256]. A current model has estimated that R5 is transmitted from male to female at one per 100 episodes of intercourse when viral RNA is present at 100 000 copies/ml of semen, versus three per 10 000 episodes when viral RNA is present at 1000 copies/ml [4]. Clearly, additional studies of co-receptor expression and control of HIV infection in genital tissues are needed.

Men and women may harbor HIV strains of different tropism in the genital tract and peripheral blood [46,59,72,170,258–260]. Parenthetically, this knowledge may be useful for studies of the reseeding of the blood compartment from the genital tract. Most studies of viral tropism in primary infection have focused on men and infants. These studies suggest that infection is limited to monotropic (CCR5) virus that evolves over time. In contrast, other studies suggest that women may be infected at the time of seroconversion with viruses of multiple tropisms [253]. It is unclear whether the presence of these multiply tropic viruses represents superinfection or infection at different sites within the genital compartment. Not enough women have been followed after acute infection to verify monotropic infection in men and infants and polytropic infection in women.

HIV subtypes, superinfection and recombination

Although clade B predominates in North America and Europe, more than one-half of all worldwide HIV infections involve clade C virus. Whereas the rapidly growing epidemic in South-east Asia is due to a recombinant AE virus, epidemics involving an AB recombinant in Eastern Europe and a BC recombinant in China are of increasing concern [261].

There is growing evidence that some HIV-1 subtypes may differ with regard to virus levels in blood, disease progression, chemokine co-receptor use, transmission and replication rates (see [262]). Differences between the blood and genital compartment for these subtypes have not been systematically studied, although clade AE differences between blood and genital fluids for men and women seem to be consistent with other published data for clade B [83,263]. Differences have been noted in clade C HIV RNA level and phenotype between blood and semen; however, these differences may reflect the effects of host and/or environmental factors such as co-infections on the shedding of HIV in the genital tract [3,150].

The presence of recombinant HIV is evidence for superinfection by two genetically distinct viruses [262]. However, the importance of the genital tract in establishing a superinfection either within a clade (intra-subtype) or between clades (inter-subtype), as a first step toward recombination, requires further study. Nevertheless, the establishment of HIV superinfection in the genital submucosa could facilitate sexual transmission and the evolution of the worldwide HIV pandemic. A recent female chimpanzee model of genital infection with subtypes B and AE showed that low-level infection was established without seroconversion after mucosal exposure to HIV [264], suggesting that the genital tract evolution of infection is testable.

HIV variation

HIV infection may evolve differently in women than in infants or men if women are infected with multiple variants [253,265]. In women, it appears that viral evolution varies between compartments, suggesting a major role for immunological pressure [253]. The existence of a similar situation between the blood and genital compartments in men has not been adequately evaluated [258].

A question that arises is the role of the genital compartment in reseeding other compartments over the course of infection. Structured treatment interruptions may provide the opportunity to assess the role of the genital tract vis-á-vis other lymphocyte compartments in reseeding the blood compartment, using drug resistance or viral phenotype as a marker.

Viral load and transmission

Two major issues confronting our understanding of HIV transmission are determining what constitutes the transmissible unit and identifying the exact genital site of infection.

We do not know whether cell-free or cell-associated virus or both are necessary for transmission. Earlier data suggested a possible difference in the male-to-female (versus female-to-male) transmission rate, with asymptomatic men transmitting five times more efficiently than asymptomatic women [266,267]. However, risk of transmission varies with stage of disease and viral plasma RNA levels [189,267]. Men and women did not differ in the rate of transmission during symptomatic infection [267]. The level of plasma viral RNA appears to be lower in women than in men early in infection, but does not differ during the later stages of the disease [268]. A more recent study in discordant couples suggests that viral load in the peripheral blood may be more predictive of transmission in women than in men [269]. However, in the Rakai, Uganda study, the main determinants of HIV-1 transmission per coital act were plasma viral RNA levels and genital ulcer disease, but not sex [270].

Clearly, much public health benefit can be achieved by therapeutically lowering the plasma RNA level. However, the genital shedding of virus is quite complex on an individual basis and cannot be assumed to be solely reflective of plasma viral RNA level and risk of transmission. The neutralizing antibody response in the host, viral load and the replicative capacity of the virus may all influence the outcome of male-to-female transmission [271].

The genital site(s) of infection in men and women will most likely differ, unless infection occurs through receptive anal intercourse. Model systems using cervicovaginal tissue have been developed to study HIV transmission in women [272–274]. Similar models are not available for men.

Numerous layers of squamous epithelial cells protect the female genital tract, except in the transition zone of the exocervix and within the endocervical canal (Fig. 2). This squamous epithelial layer is thought to be the major barrier to infection. However, the uterine contractions associated with intercourse are designed to move semen into the endocervical canal, where the columnar epithelial barrier is only a single cell thick and the basement membrane is extremely thin [275].

Some model systems have shown cell-associated X4 and R5 virus trancytosis through human endometrial cells [11]. In vivo, trancytotic virus would reach the numerous CD4+ and antigen-processing cells that inhabit the endometrial submucosa [235]. Other studies have shown that primary cervical epithelial cells cannot support the replication of cell-free or cell-associated HIV, although the virus can be sequestered for a long time in these epithelial cells [276].

More R5-expressing lymphocytes appear to be present in the vaginal submucosa than in peripheral blood, but receptor density is approximately the same [255]. Although dendritic cells in the genital tract have been shown to harbor and disseminate infectious virus in animal models [277], dentritic cells isolated from the human vagina have receptors for primarily X4 and not R5 viruses [256]. Non-productive infection of unstimulated vaginal T cells occurs with both X4 and R5 viral strains, and productive infection occurs in the presence of stable dendritic cell–T cell complexes [256]. Thus, the primary routes of infection in women are probably through disruption of the vaginal epithelial layer, infection at the level of the ecto- and endocervix and, possibly, the endometrium, with migration of virus from the mucosal site in dendritic cells to potential lymphoid sites of CD4+ cell infection [256].

The ratio of female-to-male HIV transmission in developing versus developed countries was calculated to be 341, which contrasts with the male-to-female transmission ratio of 2.9 [110]. Increased HIV transmission in developing countries may be driven by (1) increased viral shedding, which increases the female's infectiousness (as summarized in Table 2), and, perhaps more importantly, (2) the much lower rate of circumcision in developing countries, increasing male susceptibility to infection [109,110].

In men, critical determinants of infection risk include the presence of Langerhans cells in the foreskin, with extension to the ampulla or the receptive environment of the coronal sulcus [278]. However, there is a complex interplay between genital ulcerative disease, poor genital hygiene, the intact foreskin, penile mucosal abrasions and acquired heterosexual HIV infection [110,279]. A recent meta-analysis concluded that an intact foreskin increases the risk of infection between 1.43 and 1.67, depending on the analysis model [279].

Another study found that the rate of male-to-female transmission was not significantly different from the rate of female-to-male transmission (approximately 12 infections per 100 person-years) but that the incidence was higher among uncircumcised male partners (40 of 137) [189]. Although there were no seroconversions among 50 circumcised male partners (P < 0.001), these observational data may be confounded by age at circumcision [280,281], Muslim faith and the concordant seronegative status of the couples studied [282]. It is not clear what the role of circumcision should be in developing countries or if circumcision explains the lower female-to-male transmission rates in the United States, where most men are circumcised [189,283]. Prospective randomized intervention studies that control for confounding variables will be required to definitively address the role of circumcision in HIV transmission [281].

Future directions

The interactions among HIV, the host and other genital co-pathogens are more complex than was first appreciated. The study of HIV-1 in the genital tract reveals not only that there are sex differences but also that much more needs to be learned about how these differences alter the response of genital HIV infection to other STIs. Research should give attention to the normative response of changes in immunoglobulin concentration with age (pre- versus post-menopausal), sex hormones and natural genital mucosal defense mechanisms in men and women. Future studies should include a more comprehensive assessment of genital virological, microbiological, immunological and nutritional parameters to provide a complete picture of the pathogenesis of genital tract HIV-1 disease and the pathophysiology of HIV sexual transmission. In women, these parameters must be correlated with the reproductive life cycle and with histological and morphological changes in the cervix and the cervical transformation zone over this life cycle.

Table 5 summarizes several research areas that should be addressed. Future research will need to establish the sites of genital viral infection and shedding and the mechanism by which viral phenotype is selected at the genital mucosa following acute infection. Because HIV and other viral and non-viral co-pathogens probably interact at or near the mucosal surface, understanding the mechanisms of infection and shedding will depend, in part, on characterizing cell targets and their co-receptor expression and determining how hormones and the response to treatment of genital co-infections influences genital HIV shedding.

The current approach of sampling the female genital tract by performing a simple flush (CVL) with HIV RNA quantification is generally inadequate. Consideration should be given to the phases of the woman's menstrual and reproductive life cycle. Therapeutic intervention protocols with primary viral endpoints in plasma should consider adding a secondary genital viral endpoint. For example, is the protocol designed to assess genital tract cell-free or cell-associated virus, or both? At what level of the female genital tract does HIV infection occur and does susceptibility to infection vary with the menstrual cycle and phase of a woman's reproductive life cycle? How quantitative must the endpoint be? Should the time of sample collection take into consideration the woman's hormonal status and ovulatory phase? Is it important to discern the site of viral shedding (i.e., endo- versus exocervix)? CVL sampling methods must be standardized so that the effect of CVL dilution can be accounted for in defining the denominator.

Additional resources should probably not be devoted to cross-sectional or longitudinal observational studies of the association between co-pathogens and HIV infection in men and women. Instead, intervention studies are needed to establish the mechanism of the interaction between HIV and genital co-pathogens, focusing on the treatment of genital tract co-infections and the assessment of genital tract viral shedding.

Studies are needed to evaluate the potential for discordant viral evolution between the blood compartment and the genital tract. The genital submucosa may be an important site for recombination between viral variants, with subsequent seeding of these recombinants to the local lymphoid tissue. This possibility could be evaluated in the ex vivo explant and animal models of genital infection. Prospective observational studies may be required to evaluate the trafficking and co-receptor use of virus between the genital and blood compartments and the role of trafficking in reseeding different compartments. We are just beginning to understand the contribution of co-receptor use and changes in viral genotype/phenotype in the female and male genital tracts. Given technological advances in this area and the use of tissue-based models, we can expect to learn much more about HIV compartments and reservoirs over the next few years.

We do not need additional cross-sectional studies to assess the association of plasma and genital tract RNA levels. Instead, more studies are needed to evaluate the effect of prospective therapeutic interventions, or change in therapy on relative changes in plasma and genital tract viral load. These studies must consider the impact of genital tract co-infections on the pathophysiology of HIV infection and transmission in men and women.

At the time of this review, there was only limited peer-reviewed data on antiretroviral drug penetration into the female genital tract. However, such data are critical for studying the development of viral resistance, viral evolution and reseeding of virus from the genital tract to other compartments.

A growing body of research is just beginning to consider the impact of menstrual hormone fluctuation on immunity and natural defenses against HIV infection. Similar types of studies in men should not be overlooked.

Certain factors that may influence HIV shedding in the male and female genital tracts (sex hormones and circumcision) should be evaluated with regard to their impact on transmissibility and infectability.

In conclusion, prevention modalities that include microbicides, vaccines and ART provide a unique opportunity to increase our understanding of the pathogenesis of HIV in the genital tracts of men and women. Assessment of viral and immunological parameters and associated co-variables will be essential for understanding the success or failure of these prevention methodologies. In addition, such studies will be important for the development of effective assisted reproductive technologies for HIV-discordant couples. Pathogenesis should be incorporated as an integral component of prevention science.

Acknowledgements

We would like to thank Susan Cu-Uvin, MD, Brown University, and Trent MacKay, MD, National Institute of Child Health and Human Development, for their thoughtful comments and helpful suggestions. We also thank Charles Muller, PhD, University of Washington, and Jonathan Cohn, MD, Wayne State University School of Medicine, for sharing unpublished data, and Margaret Camarca, WESTAT, Inc., for technical assistance.

Sponsorship: This study was supported in part by NIH HD 40540-02, cooperative agreement AI-27664 and the University of Washington Center for AIDS Research AI-30731 (RWC); NIH HD-40541-02 (ALL); NIH contract HD-33162.